Viruses in the family Poxviridae, including vaccinia virus (VACV) and variola virus, are characterized by a large linear double-stranded DNA genome (130-300 kb) packaged in a relatively large virion (xcx9c350xc3x97270 nm), and a cytoplasmic site of replication (reviewed by Moss, 1996, In xe2x80x9cFields Virologyxe2x80x9d, D. M. Knipe et al. Eds., vol. 3, pp 2637-2671. Lippincott-Raven, Philadelphia). Assembly of VACV virions begins with condensation of dense granular material into membrane-wrapped particles called intracellular mature virions (IMV). Recent findings indiate the IMV are wrapped by a single membrane (Hollingshead et al., 1999, J. Virol. 73, 1503-1517) rather than a double membrane as previously reported. IMV are then enveloped in two additional membranes derived from the trans Golgi to form multiple membrane-warpped particles called intracellular enveloped virions (IEV) (Schmelz et al., 1994, J. Virol. 68, 130-147). IEV are moved, possibly by actin polymerization (Cudmore et al., 1995, Nature 378, 636-638), to the cell periphery, where the outermost membrane fuses with the cell plasma membrane, exposing a cell-associated eneveloped virion (CEV) (Blasco and Moss, 1991, J. Virol. 65, 5910-5920). CEV are released from the cell as extracellular enveloped virions (EEV), which play a role in long-range spread of the virus (Payne, 1980, J. Gen. Virol. 50, 89-100). IMV released from disrupted cells and EEV are both infectious forms of VACV.
The primary therapeutic tool for the control and eradication of infection with VACV include a live virus vaccine to prevent disease, and a vaccinia immune globulin (VIG) to treat dissminated infections. The existing VIG product is derived from human donors who have been vaccinated with the smallpox vaccine, vaccinia virus. As with all human products, the existing VIG must be tested exhaustively for blood borne human pathogens such as human immunodeficiency virus and hepatitus B. Therefore, the existing VIG suffers from several drawbacks including the necessity for using human volunteers, i.e. the use of a live virus as an immunogen which could cause infectious lesions that scar in healthy individuals and severe disseminated life-threatening infection in immunocompromised individuals. And, despite continuous screening of the donor population to assure consistency which is very expensive, product lots can vary significantly between batches and geographic regions.
Therefore, there is a need to provide an immune globulin composition which is safe and precisely defined, and which does not rely on human donors. However, it is not known which components of the vaccinia VIG are important for protection nor how many of the xcx9c200 genes contained in the vaccinia genome encode proteins that would elicit a protective response upon passive transfer of monoclonal antibodies directed to such proteins.
This application satisfies the need mentioned above. This application describes a vaccinia immunoglobulin composition which can serve as a replacement for the presently used VIG. The vaccinia immunoglobulin composition of the present invention is composed of one or more monoclonal antibody against vaccinia antigens defined to be important for protection. To identify potential targets for poxvirus therapeutics, we generated and characterized a panel of 400 VACV-specific monoclonal antibodies (MAbs) in mice. The monoclonal antibodies were first tested for their ability to neutralize virus and then were tested for their ability to protect mice from challenge. Two challenge models were used, one that involves dissemination of the virus (in suckling mice) and a challenge that involves a massive challenge dose (by intraperitoneal injection). To our surprise, the ability of the MAbs to inhibit plaque formation by vaccinia virus, a standard assay of virus neutralization, did not always predict their protective efficacy. Moreover, the monoclonal antibodies differed in their ability to provide protection depending on the challenge model.
We found that the majority of moderately neutralizing monoclonal antibodies were directed against a 34 KDa protein later determined to be D8L which on its own did not provide protection in mice. Another monoclonal antibody which was neutralizing did not protect against challenge when given alone to mice and was directed against the protein A27L. Neutralizing MAbs binding to the 29-KDa protein (e.g. MAb-10F5, and MAB-7D11), protected mice against intraperitoneal challenge and were found to react with the IMV product of the L1R gene first described in Wolffe, E. J. et al., 1995, Virology 211, 53-63). Nonneutralizing MAbs binding to 23 to 28-kDA protein (e.g. MAb-1G10) protected against challenge with VACV (strain WR) in suckling mice. The target of MAb-1G10 was the EEV product of the A33R gene (Roper et al., 1996, J. Virol. 70, 3753-3762).
The LIR and A33R gene product will be called L1R and A33R, respectively. L1R is an essential myristoylated protein associated with the IMV membrane and is thought to play a role in IMV attachment or penetration (Franke et al., 1990, J. Virol. 64, 5988-5996; Ravanello et al., 1993, J. Gen. Virol. 75, 1479-1483; Ichihashi et al., 1994, Virology 202, 834-843; Ravanello and Hruby, 1994, J. Gen. Virol. 75, 1479-1483; Wolffe et al., 1995, supra). A33R is a nominally nonessential glycosylated/palmitated protein that forms dimers and is incorporated into the outer membrane of EEV (Payne, 1992, Virology 187, 251-260; Roper et al., 1996, supra). A33R is thought to be involved in facilitating direct cell-to-cell spread via actin-containing microvilli (Roper et al., 1998, J. Virol. 72, 4192-4204). Homologs of L1R and A33R are present in other Orthopoxviruses, e.g. between VACV and variola, L1R identity is 99.6% and A33R is 94.1% (Massung et al., 1994, Virology 201, 215-240).
Therefore, it is an object of the present invention to provide a composition of one or more monoclonal antibody directed against at least one, preferably two or more, vaccinia virus antigens. Antigens preferably include L1R and A33R.
It is another object of the present invention to provide monoclonal antibodies which protect against vaccinia virus infection and bind to epitopes on L1R and A33R gene products. The monoclonal antibodies described below recognize epitopes on the VACV strain Connaught L1R sequence (Genebank #Af226617) and the Connaught strain A33R gene sequence (Genebank #Af226618). L1R and A33R homologs from other poxviruses can be used as immunogens to produce monoclonal antibodies which would most likely be protective since the homologs in other poxviruses have high identity with the VACV proteins. Other poxviruses include other Orthopoxviruses such as variola virus, monkeypox virus, cowpox virus, Parapoxviruses such as orf virus, paravaccinia virus, and unclassified poxviruses such as Tanapoxvirus, Yabapoxvirus and Molluscum contagiosum.
It is yet another object of the present invention to provide a composition comprising humanized monoclonal antibodies of the present invention for example anti-L1R antibody, or anti-A33R antibody or a mixture thereof, as a vaccinia immunoglobulin replacement. The vaccinia immunoglobulin replacement may further contain other antibodies specific for vaccinia antigens shown to be effective for eliciting neutralizing/protective antibodies, for example H3L, D8L, B5R, A27L, and A17L. In addition, MAbs against L1R and A33R homologs from other poxviruses can be used alone or in combination with the vaccinia MAbs to provide a therapeutic and prophylactic composition.
It is another object of the invention to provide for antibodies that are functionally equivalent to the antibodies listed above. These functionally equivalent antibodies substantially share at least one major functional property with an antibody listed above and herein described comprising: binding specificity to L1R and A33R, immunoreactivity in vitro, protection against vaccinia challenge when administered prophylactically or therapeutically, competition for same binding site on L1R and A33R. The antibodies can be of any class such as IgG, IgM, or IgA or any subclass such as IgG1, IgG2a, and other subclasses known in the art. Further, the antibodies can be produced by any method, such as phage display, or produced in any organism or cell line, including bacteria, insect, mammal or other type of cell or cell line which produces antibodies with desired characteristics, such as humanized antibodies. The antibodies can also be formed by combining an Fab portion and a Fc region from different species.
It is another object of the present invention to provide for mixtures of antibodies according to the present invention, as well as to methods of using individual antibodies, or mixtures thereof for the prevention and/or therapeutic treatment of vaccinia virus infections in vitro and in vivo, and/or for improved detection of vaccinia infections.
It is yet another object of the present invention to treat or prevent vaccinia virus infection by administering a therapeutically or prophylactically effective amount of one antibody of the present invention or a mixture of antibodies of the present invention to a subject in need of such treatment.
It is another object of the present invention to provide passive vaccines for treating or preventing vaccinia virus infections comprising a therapeutically or prophylactically effective amount of the antibodies of the present invention which protect against vaccinia virus, in combination with a pharmaceutically acceptable carrier or excipient.
It is yet another object of the present invention to provide a method for diagnosis of vaccinia virus infection by assaying for the presence of vaccinia in a sample using the antibodies of the present invention.
It is still another object of the present invention to provide novel immunoprobes and test kits for detection of vaccinia virus infection comprising antibodies according to the present invention. For immunoprobes, the antibodies are directly or indirectly attached to a suitable reporter molecule, e.g., and enzyme or a radionuclide. The test kit includes a container holding one or more antibodies according to the present invention and instructions for using the antibodies for the purpose of binding to vaccinia virus to form an immunological complex and detecting the formation of the immunological complex such that presence or absence of the immunological complex correlates with presence or absence of vaccinia virus.
It is another object of the present invention to provide anti-idiotypic antibodies raised against one of the present monoclonal antibodies for use as a vaccine to elicit an active anti-vaccinia response.